title: A novel calcium-binding site of von Willebrand factor A2 domain regulates its cleavage by ADAMTS13.
abstract: The proteolysis of VWF by ADAMTS13 is an essential step in the regulation of its hemostatic and thrombogenic potential. The cleavage occurs at strand β4 in the structural core of the A2 domain of VWF, so unfolding of the A2 domain is a prerequisite for cleavage. In the present study, we present the crystal structure of an engineered A2 domain that exhibits a significant difference in the α3-β4 loop compared with the previously reported structure of wild-type A2. Intriguingly, a metal ion was detected at a site formed mainly by the C-terminal region of the α3-β4 loop that was later identified as Ca(²+) after various biophysical and biochemical studies. Force-probe molecular dynamic simulations of a modeled structure of the wild-type A2 featuring the discovered Ca(²+)-binding site revealed that an increase in force was needed to unfold strand β4 when Ca(²+) was bound. Cleavage assays consistently demonstrated that Ca(²+) binding stabilized the A2 domain and impeded its unfolding, and consequently protected it from cleavage by ADAMTS13. We have revealed a novel Ca(²+)-binding site at the A2 domain of VWF and demonstrated a relationship between Ca(²+) and force in the regulation of VWF and primary hemostasis.
authors.affiliation: State Key Laboratory of Molecular Biology and Research Center for Structural Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Minyun
authors.initials: M
authors.lastname: Zhou
authors.forename: Xianchi
authors.initials: X
authors.lastname: Dong
authors.forename: Carsten
authors.initials: C
authors.lastname: Baldauf
authors.forename: Hua
authors.initials: H
authors.lastname: Chen
authors.forename: Yanfeng
authors.initials: Y
authors.lastname: Zhou
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Springer
authors.forename: Xinping
authors.initials: X
authors.lastname: Luo
authors.forename: Chen
authors.initials: C
authors.lastname: Zhong
authors.forename: Frauke
authors.initials: F
authors.lastname: Gräter
authors.forename: Jianping
authors.initials: J
authors.lastname: Ding
mesh_terms: D051722:ADAM Proteins / Q000378:metabolism*; D000071120:ADAMTS13 Protein; D001665:Binding Sites / Q000502:physiology; D014408:Biomarkers, Tumor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D057809:HEK293 Cells; D006487:Hemostasis / Q000502:physiology*; D006801:Humans; D016296:Mutagenesis; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D013927:Thrombosis / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D014408:Biomarkers, Tumor; D002135:Calcium-Binding Proteins; D014157:Transcription Factors; C496188:VWA2 protein, human; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human; D002118:Calcium